Spleens were mechanically processed in 2 mL of Cell Staining Buffer (BioLegend) over a 40‐mm strainer, and 30 µL was used for staining. Cell Staining Buffer was added to achieve a final volume of 100 µL. The following Abs (PE‐rat CD4, BioLegend, USA) in a total volume of 50 µL were added at the concentrations suggested by the manufacturer. Isotype control Abs were used at the same protein concentrations as the corresponding marker‐specific Abs. Cells were stained in the dark on ice for 20 min, and 1.6 mL of One‐Step Fix/Lyse Solution (eBioscience) was added, mixed, and incubated with the cells for 30 min at room temperature. The cell solution was centrifuged, and the supernatant was discarded. The cells were washed twice with Cell Staining Buffer, resuspended in 2 mL of Cell Staining Buffer, sorted on a FACSCanto II (Becton Dickinson Biosciences), and analyzed using Flow Jo V10.0.7.
One step fix lyse solution
Manufactured by Thermo Fisher Scientific
One‐Step Fix/Lyse Solution is a laboratory reagent designed to fix and lyse cells in a single step, preparing them for further analysis. It is a versatile tool for sample preparation in various applications that require cell fixation and lysis.
Automatically generated - may contain errors
2 protocols using one step fix lyse solution
1
Phenotyping Murine Splenic T Cells
2
Single-Cell Flow Cytometry Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cell pellets were resuspended in 2% FBS–PBS and incubated with streptokinase for 10 min shaking at 37 °C; 100 μL were removed, washed with PBS, and resuspended in 1:100 fixable viability dye efluor450 (eBioscience) in PBS. Cells were incubated at 4 °C for 30 min protected from light. Cells were washed two times with 2% FBS–PBS. Red blood cells were then lysed, and the sample was fixed with one-step fix/lyse solution (eBioscience) following the protocol. Cells were run on a CytoFlex analyzer (Beckman Coulter). Analysis was performed using FlowJo software v10.
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